Wheat gluten peptide as well as preparation method and related application thereof

Abstract

The invention discloses vital gluten peptide as well as a preparation method and related application thereof, and relates to the field of biology. The amino acid sequence of the wheat gluten peptide provided by the embodiment of the invention is selected from any one or more of LK, LLL, LLW, LPW, WLD, WLE and WVP, the wheat gluten peptide has the effect of remarkably enhancing the salt stress resistance of yeast, and when the wheat gluten peptide is applied to industrial fermentation, the environmental stress resistance of the yeast can be effectively enhanced, and the yield of fermented products is increased; under the condition of high salt stress, yeast proliferation can be effectively promoted, yeast cell membrane integrity and mitochondrial membrane potential are maintained, and the content of active oxygen in cells is reduced. The wheat gluten peptide provides a new way for enhancing the resistance of yeast to salt stress, and can be widely applied to yeast fermentation.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to gluten peptides, a preparation method thereof and related applications. Background Art

[0002] Existing technologies mostly use high-tolerance strain selection and exogenous nutrient supplementation to enhance the resistance of Saccharomyces cerevisiae to salt stress. However, the genetic engineering method for breeding high-tolerance yeast strains is difficult to achieve industrial application due to potential transgenic food safety issues; exogenous nutrient supplementation (especially nitrogen source) is considered a method to improve yeast tolerance. At present, there is a lack of exogenous nutrients that can safely and efficiently enhance the resistance of Saccharomyces cerevisiae to salt stress.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide gluten peptide and a preparation method and related applications thereof.

[0005] The present invention is achieved in that:

[0006] In a first aspect, an embodiment of the present invention provides a polypeptide, whose amino acid sequence is selected from any one of LK, LLL, LLW, LPW, WLD, WLE and WVP.

[0007] In a second aspect, an embodiment of the present invention provides a polypeptide composition, comprising: at least two of polypeptides 1 to 7; the amino acid sequences of polypeptides 1 to 7 are LK, LLL, LLW, LPW, WLD, WLE and WVP, respectively.

[0008] In a third aspect, an embodiment of the present invention provides a method for preparing a gluten hydrolyzate, comprising: filtering the product of enzymatic hydrolysis of gluten in aqueous solution to obtain components with a molecular weight of less than 1KD; and separating and purifying the components with a molecular weight of less than 1KD using a C18 chromatography column.

[0009] In a fourth aspect, an embodiment of the present invention provides a composition, the active ingredient of which includes: the polypeptide described in the above embodiment or the polypeptide composition described in the above embodiment or the gluten hydrolyzate prepared by the preparation method described in the above embodiment.

[0010] In a fifth aspect, embodiments of the present invention provide the use of the polypeptide described in the preceding embodiments, or the polypeptide composition described in the preceding embodiments, or the gluten hydrolyzate prepared by the preparation method described in the preceding embodiments in yeast fermentation or preparation of yeast fermentation products.

[0011] In a sixth aspect, embodiments of the present invention provide the use of the polypeptide described in the preceding embodiments, or the polypeptide composition described in the preceding embodiments, or the gluten hydrolyzate prepared by the preparation method described in the preceding embodiments in enhancing the resistance of yeast cells to salt stress or in preparing products that enhance the resistance of yeast cells to salt stress.

[0012] The present invention has the following beneficial effects:

[0013] The gluten peptide provided in the embodiment of the present invention has the effect of significantly enhancing yeast's resistance to salt stress. When applied to industrial fermentation, it can effectively enhance yeast's ability to resist environmental pressure and increase the yield of fermentation products. Under high salt stress conditions, it can effectively promote yeast proliferation, maintain yeast cell membrane integrity and mitochondrial membrane potential, and reduce intracellular reactive oxygen content. The gluten peptide provides a new way to enhance yeast's resistance to salt stress and can be widely used in yeast fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 To investigate the effects of adding gluten hydrolysate and its ultrafiltration fractions to the medium containing 0.8 M sodium chloride on yeast growth;

[0016] Figure 2 To study the effect of adding ultrafiltration fractions with molecular weight <1KD and their C18 fractions on yeast growth in a medium containing 0.8M sodium chloride;

[0017] Figure 3 The effect of gluten peptide on yeast growth in medium containing 0.8M sodium chloride;

[0018] Figure 4 The effect of gluten peptide addition on yeast cell membrane integrity in medium containing 0.8M sodium chloride;

[0019] Figure 5 The effect of gluten peptide addition on the intracellular ROS level of yeast in the medium containing 0.8M NaCl;

[0020] Figure 6 The effect of gluten peptide addition on yeast mitochondrial membrane potential in medium containing 0.8 M NaCl. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0022] On the one hand, an embodiment of the present invention provides a polypeptide whose amino acid sequence is selected from any one of LK (Leu-Lys), LLL (Leu-Leu-Leu), LLW (Leu-Leu-Trp), LPW (Leu-Pro-Trp), WLD (Trp-Leu-Asp), WLE (Trp-Leu-Glu) and WVP (Trp-Val-Leu).

[0023] The polypeptide provided in the embodiment of the present invention has the effect of improving yeast resistance to salt stress. When applied to industrial fermentation, it can enhance the ability of yeast to resist environmental pressure. Under high salt stress, it can effectively promote yeast proliferation, maintain yeast cell membrane integrity and mitochondrial membrane potential, and reduce the content of intracellular reactive oxygen, thereby increasing the yield of fermentation products.

[0024] On the other hand, an embodiment of the present invention provides a polypeptide composition, comprising: at least two of polypeptides 1 to 7; the amino acid sequences of polypeptides 1 to 7 are LK, LLL, LLW, LPW, WLD, WLE and WVP, respectively.

[0025] In some embodiments, the polypeptide composition comprises WVP and LPW.

[0026] In some embodiments, the polypeptide composition comprises: LLW, LPW, WLD, WLE and WVP.

[0027] On the other hand, an embodiment of the present invention provides a method for preparing a gluten hydrolysate, comprising:

[0028] Filtering the product of the enzymatic hydrolysis of the aqueous solution of gluten to obtain a component with a molecular weight of less than 1KD;

[0029] A C18 chromatography column was used to separate and purify components with a molecular weight of less than 1KD.

[0030] In some embodiments, the step of filtering the product of enzymatic hydrolysis of the aqueous solution of gluten comprises: filtering the product of enzymatic hydrolysis of the aqueous solution of gluten using an ultrafiltration membrane, and when the molecular weight of the ultrafiltration membrane is 1kD, separating and obtaining components with a molecular weight of less than 1KD.

[0031] In some embodiments, after obtaining the component with a molecular weight of less than 1KD and before column chromatography, the preparation method further comprises: freeze-drying the component with a molecular weight of less than 1KD to obtain a lyophilized powder with a molecular weight of less than 1KD. The lyophilized powder with a molecular weight of less than 1KD is dissolved and then purified and separated by a C18 chromatography column.

[0032] In some embodiments, when performing separation and purification, the eluent used is 60% to 80% ethanol water by volume, and the flow rate is 0.5 to 2 mL / min. The volume fraction of ethanol water can be any one of 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, or any two thereof. The flow rate can be any one of 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2 mL / min, or any two thereof.

[0033] In some embodiments, when separation and purification are performed, the preparation method further comprises: collecting any one or more of fraction 1, fraction 2, fraction 3 and fraction 4; wherein the peak time of fraction 1 is 30 to 36 min, specifically any one of 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36 min or a range between any two thereof; the peak time of fraction 2 is 61 to 67 min, specifically 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65, 65.5, 66, 66.5, 67.5, 68.5, 69.5, 70.5, 71.5, 72.5, 73.5, 74.5, 75.5, 76.5, 77.5, 78.5, 79.5, 80.5, 81.5, 82.5, 83.5 7min, any one or a range between any two of the above; the peak time of the fraction 3 is 81-87min, specifically 81, 81.5, 82, 82.4, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87min, or a range between any two of the above; the peak time of the fraction 4 is 113-119min, specifically 113, 113.5, 114, 114.5, 115, 115.5, 116, 116.5, 117, 117.5, 118, 118.5, 119min, or a range between any two of the above.

[0034] In some embodiments, after collecting any one or more of fractions 1 to 4, the preparation method further comprises concentrating the collected fractions by vacuum evaporation and freeze-drying to obtain gluten peptides.

[0035] In some embodiments, the step of obtaining the product after enzymatic hydrolysis of the aqueous solution of gluten comprises:

[0036] heating the aqueous gluten solution homogenized by ultra-high pressure to obtain a heated feed solution;

[0037] The pH of the feed solution is adjusted to 6.0-9.0, and protease is added to perform enzymolysis to obtain an enzymolysis product;

[0038] The enzymatic hydrolysis product is filtered, and the precipitate is removed to obtain the supernatant.

[0039] In some embodiments, the mass concentration of gluten in the gluten aqueous solution is 5% to 15%, which can be any one of 5%, 6%, 8%, 10%, 12%, 14%, 15%, or a range between any two of them.

[0040] In some embodiments, the ultra-high pressure homogenization conditions are: pressure 100-300 MPa, time 10-30 min. The pressure can be any one of 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300 MPa or a range between any two. The time can be any one of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 min or a range between any two.

[0041] In some embodiments, the heating is heating the gluten aqueous solution to 40-60° C. The temperature can be any one of 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60° C. or a range between any two thereof.

[0042] In some embodiments, the pH of the slurry is adjusted to a pH between 6.0 and 9.0, which may specifically be any one of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, or a range between any two of them.

[0043] In some embodiments, the protease comprises one of alkaline protease, neutral protease, composite protease, trypsin and papain.

[0044] In some embodiments, the amount of the protease added is 0.5% to 1.5% of the mass of the gluten powder, and the amount added can specifically be any one of 0.5%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.5% or a range between any two of the mass of the gluten powder.

[0045] In some embodiments, the conditions of the enzymatic hydrolysis include: 40-60°C, 10-20h. The temperature can be any one of 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60°C or a range between any two thereof; the time can be any one of 10, 12, 14, 16, 18, 20h or a range between any two thereof.

[0046] In some embodiments, after the enzymolysis is completed and before filtering, the preparation method further comprises: deactivating the protease of the enzymolysis product and cooling it to room temperature. The conditions for deactivating the protease include: 95-100°C for 10-20 minutes. The temperature can be any one of 95, 96, 97, 98, 99, 100°C or a range between any two of them. The time can be any one of 10, 12, 14, 16, 18, 20 minutes or a range between any two of them.

[0047] In some embodiments, the step of filtering the enzymatic hydrolysis product and removing the precipitate comprises: centrifuging the enzymatic hydrolysis product at a speed of 4000-6000 rpm for 5-15 min. The speed can be any one of 4000, 4500, 5000, 5500, 6000 rpm or a range between any two of them, and the time can be any one of 5, 6, 8, 10, 12, 14, 15 min or a range between any two of them.

[0048] The preparation method provided by the embodiment of the present invention uses gluten as a raw material, adopts an ultra-high pressure homogenization technology to pre-treat the gluten, and then uses a restricted controllable enzymatic hydrolysis technology to degrade the gluten so that the active polypeptides can be fully released, and uses an ultrafiltration separation technology in combination with a C18 column to separate and enrich the gluten active peptides, thereby achieving efficient enrichment of the gluten active peptides. The method has the advantages of simple operation, green environmental protection, high extraction efficiency, and can prepare gluten peptides with the function of enhancing yeast resistance to salt stress at low cost and high efficiency.

[0049] On the other hand, an embodiment of the present invention provides a composition, the active ingredient of which includes: the polypeptide described in the above embodiment or the polypeptide composition described in any of the above embodiments or the gluten extract prepared by the preparation method described in any of the above embodiments.

[0050] In some embodiments, the composition further comprises: Saccharomyces cerevisiae fermentation feedstock.

[0051] On the other hand, an embodiment of the present invention provides the use of a gluten extract prepared by the polypeptide described in any of the preceding embodiments, or the polypeptide composition described in any of the preceding embodiments, or the preparation method described in any of the preceding embodiments in yeast fermentation or preparation of yeast fermentation products.

[0052] In some embodiments, the yeast comprises Saccharomyces cerevisiae.

[0053] In some embodiments, the yeast fermentation product includes any one or more of kimchi, bean paste, soy sauce, bread, and fermented feed.

[0054] In addition, an embodiment of the present invention provides the use of a gluten extract prepared by the polypeptide described in any of the preceding embodiments, or the polypeptide composition described in any of the preceding embodiments, or the preparation method described in any of the preceding embodiments in enhancing the resistance of yeast cells to salt stress or in preparing a product that enhances the resistance of yeast cells to salt stress.

[0055] In some embodiments, the yeast is the same as described in any of the above embodiments. The features and performances of the present invention are further described in detail below in conjunction with the embodiments.

[0056] Example 1

[0057] 1. Experimental Materials

[0058] YNB medium: Prepared by dissolving 2 g glucose and 6.7 g YNB in ​​1000 mL water;

[0059] Salt (sodium chloride) stress medium: Sodium chloride (final concentration of sodium chloride in the medium is 0.8 M) was added to YNB medium.

[0060] Brewer's yeast: Saccharomyces pastorianus, was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on December 24, 2010, with the deposit number of CGMCC No.4466.

[0061] 2. Gluten hydrolysis

[0062] Wheat gluten powder was dispersed in distilled water at a concentration of 0.1 kg / L and homogenized by ultra-high pressure (100 MPa, homogenization for 30 min). The mixed solution was then heated to 50°C in a water bath, and the pH of the solution was adjusted to 9.0 with a sodium hydroxide solution (0.1 mol / L). 1.5% (w / v) trypsin was added to the solution for 1 day of enzymolysis at 50°C constant temperature stirring, and then the enzymolysis solution was heated to 100°C to kill the enzyme. After the enzymolysis solution was cooled, the supernatant was collected (centrifuged after cooling, the centrifugation conditions were 4°C, 10000 g, 15 min), and an enzymolysis solution was obtained. The enzymolysis solution was freeze-dried to obtain a hydrolyzate which was stored in a -20°C refrigerator.

[0063] 3. Ultrafiltration separation and activity analysis of gluten hydrolysate

[0064] The prepared hydrolyzate was prepared into a solution with a substrate concentration of 4‰ using deionized water, and separated using a 3KD ultrafiltration membrane through an ultrafiltration device to obtain a retained component of >3KD and a permeated component of <3KD. The permeated component of <3KD was then separated using a 1KD ultrafiltration membrane to obtain a retained component of 1-3KD and a permeated component of <1KD. The components were then concentrated and collected, vacuum freeze-dried, and stored at -20°C for future use.

[0065] 0.15 g / mL of hydrolyzate, >3 KD, 1-3 KD, and <1 KD were added to 0.8 M salt stress YNB medium, and then the starting OD 600 Yeast seeds with a density of about 0.1 were placed in a shaker at 25°C and samples were taken at regular intervals. The OD of yeast samples under salt stress conditions was recorded by observation. 600 The effects of gluten hydrolysate and its ultrafiltration fraction on cell growth were analyzed.

[0066] Figure 1 The growth of yeast cells in each component under 0.8M salt stress. From the experimental results, it can be seen that the ultrafiltration components with a molecular weight of <1KD in the ultrafiltration components can significantly improve the salt stress tolerance of Saccharomyces cerevisiae to varying degrees. The cell growth of gluten hydrolysate and its ultrafiltration components was significantly higher than that of the blank group (p<0.05), and the yeast cells of each component entered the logarithmic phase at 12h, among which the yeast cells of the ultrafiltration components with a molecular weight of <1KD showed the best growth rate.

[0067] 4. C18 column separation and purification of components with molecular weight <1KD

[0068] The <1KD component was separated using a 12mm×100cm pressure chromatography column filled with SMB-1800-20 / 45C18 filler. The eluent was 70% (v / v) ethanol. The flow rate was 1mL / min, and four separated components were prepared. They were named Fr1 (33.46min), Fr2 (64.18min), Fr3 (84.53min), and Fr4 (116.64min) according to the peak time. The components were concentrated and lyophilized and stored in a -20°C refrigerator.

[0069] Under 0.8M salt stress, the ultrafiltration fraction with molecular weight <1KD and its C18 separation fraction were added to the culture medium to test the effect of each fraction on yeast cell growth ( Figure 2 ). In 0.8M salt stress medium, each fraction improved the osmotic pressure tolerance of Saccharomyces cerevisiae and promoted the proliferation of yeast cells, among which Fr3>Fr4>molecular weight<1KD>Fr2>Fr1>control. This result shows that C18 fraction Fr3 is enriched with more active substances that are beneficial to the growth of yeast under salt stress.

[0070] Studies have shown that most yeast cells can only transport and utilize small peptides, and that yeast cells have a related transport system for the absorption and transport of peptides, among which the PTR transport system mainly transports dipeptides and tripeptides. The amino acid sequence of small peptides in the highly active component Fr3 was identified using OrbitrapElite LC-MS / MS, and 7 dipeptides and tripeptides were obtained: LK (Leu-Lys), LLL (Leu-Leu-Leu), LLW (Leu-Leu-Trp), LPW (Leu-Pro-Trp), WLD (Trp-Leu-Asp), WLE (Trp-Leu-Glu) and WVP (Trp-Val-Leu).

[0071] Seven small peptides (LK, LLL, LLW, LPW, WLD, WLE and WVP) were selected for activity analysis. The effects of the addition of seven small peptides on the ability of yeast cells to resist salt stress were analyzed in YNB medium containing 0.8M sodium chloride. The results are as follows Figure 3 As shown. Under 0.8M sodium chloride stress, the growth ability of yeast cells supplemented with 7 small peptides was significantly higher than that of the control group, among which yeast supplemented with LPW and WVP showed a higher growth rate. These results show that under salt stress conditions, the 7 gluten peptides can promote the proliferation of yeast cells, further indicating that the supplementation of 7 small peptides (especially LPW and WVP) can enhance the resistance of yeast cells to salt stress.

[0072] 5. Effects of gluten peptides on yeast cell membrane integrity, ROS and intracellular mitochondrial membrane potential

[0073] The integrity of the yeast cell membrane was detected by propidium iodide (PI). The yeast cells under the experiment were collected by centrifugation, washed three times with PBS, and then suspended with PBS to a cell density of about 6Lg CFU / mL. The PI probe was added to a final concentration of 15mM, and then incubated at 25°C in the dark for 20min. The fluorescence intensity was then detected using a multifunctional microplate reader. The detection wavelength was λex=535nm, λem=615nm.

[0074] The DCFH-DA fluorescent probe staining method was used to determine the intracellular ROS content. The yeast cells were collected by centrifugation, washed three times with PBS, and then suspended with PBS to a cell density of about 6Lg CFU / mL. The DCFH-DA probe was added to a final concentration of 10mM and incubated at 30°C in the dark for 30min. After the incubation, the cells were washed three times with PBS solution. The fluorescence intensity was then detected using a multifunctional microplate reader. The detection wavelength was λex=488, λem=525nm.

[0075] Tetramethylrhodamine ethyl ester (TMRE) was used to detect changes in mitochondrial membrane potential in yeast cells. Yeast cells were collected by centrifugation, washed three times with PBS, and suspended with PBS to a cell density of about 6Lg CFU / mL. TMRE was added at a final concentration of 100nM and incubated at room temperature in the dark for 30 minutes. After incubation, the cells were washed three times with PBS. After suspension in PBS, the fluorescence intensity was detected using a multifunctional microplate reader. The detection wavelengths were λex=549nm, λem=574nm.

[0076] The effects of seven gluten peptides (LK, LLL, LLW, LPW, WLD, WLE and WVP) on the cell membrane integrity of Saccharomyces cerevisiae under 0.8M NaCl stress were studied using PI fluorescent dye. Figure 4 As shown in the figure, the fluorescence intensity of yeast cells in the seven small peptide addition groups was reduced compared with the control group, among which LLW, LPW, WLD, WLE and WVP decreased significantly, especially WVP decreased by 47.57%, followed by LPW decreased by 45.62%. It can be seen that the supplementation of gluten peptides (especially WVP and LPW) significantly improved the integrity of cell membranes, thereby helping cells resist salt stress.

[0077] The effects of adding seven gluten peptides (LK, LLL, LLW, LPW, WLD, WLE and WVP) on the intracellular ROS level of yeast under salt stress were tested. Figure 5 . Under 0.8M sodium chloride stress, compared with the control group, the supplementation of 7 gluten peptides significantly reduced the level of ROS in yeast cells (p<0.05), among which the accumulation of ROS in yeast cells supplemented with WVP was the lowest at 35.68%, followed by LPW at 39.07%, which were reduced by 64.32% and 60.93% respectively compared with the control. This result shows that the supplementation of 7 gluten peptides (especially WVP and LPW) under salt stress can reduce the accumulation of intracellular ROS, thereby reducing the oxidative damage of yeast cells and further enhancing the resistance of yeast cells to salt stress.

[0078] The effects of seven gluten peptides (LK, LLL, LLW, LPW, WLD, WLE and WVP) supplementation on mitochondrial membrane potential of yeast cells under salt stress were determined. Figure 6 As shown. Under 0.8M sodium chloride stress, the addition of 7 gluten peptides significantly increased the fluorescence intensity of yeast cells compared with the control (p < 0.05), among which WVP showed the highest intensity of 144.56%, followed by LPW at 143.39%. These results show that under high salt stress, gluten peptides help maintain the mitochondrial membrane potential of yeast cells, thereby maintaining the stability of mitochondria and further enhancing the resistance of yeast cells to salt stress.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that Its amino acid sequence is selected from any one of LK, LLL, LLW, LPW, WLD, WLE and WVP.

2. A polypeptide composition, characterized in that: It comprises: at least two of polypeptides 1 to 7; the amino acid sequences of polypeptides 1 to 7 are LK, LLL, LLW, LPW, WLD, WLE and WVP, respectively.

3. The polypeptide composition according to claim 2, characterized in that: The polypeptide composition includes WVP and LPW; Optionally, the polypeptide composition comprises: LLW, LPW, WLD, WLE and WVP.

4. A method for preparing gluten hydrolysate, characterized in that: It includes: Filtering the product of the enzymatic hydrolysis of the aqueous solution of gluten to obtain a component with a molecular weight of less than 1KD; A C18 chromatography column was used to separate and purify components with a molecular weight of less than 1KD.

5. The preparation method according to claim 4, characterized in that: When performing separation and purification, the eluent used is 60% to 80% ethanol water by volume, and the flow rate is 0.5 to 2 mL / min; Optionally, when performing separation and purification, the preparation method further includes: collecting any one or more of fraction 1, fraction 2, fraction 3 and fraction 4; wherein the peak time of fraction 1 is 30 to 36 min, the peak time of fraction 2 is 61 to 67 min, the peak time of fraction 3 is 81 to 87 min, and the peak time of fraction 4 is 113 to 119 min.

6. The preparation method according to claim 5, characterized in that: The peak time of the fraction 1 is 32 to 34 minutes, the peak time of the fraction 2 is 63 to 65 minutes, the peak time of the fraction 3 is 83 to 85 minutes, and the peak time of the fraction 4 is 115 to 117 minutes.

7. The preparation method according to any one of claims 4 to 6, characterized in that: The step of obtaining the product after enzymatic hydrolysis of the aqueous solution of gluten powder comprises: heating the aqueous gluten solution homogenized by ultra-high pressure to obtain a heated feed solution; The pH of the feed solution is adjusted to 6.0-9.0, and protease is added to perform enzymolysis to obtain an enzymolysis product; The enzymatic hydrolysis product is filtered to remove the precipitate to obtain a supernatant; Optionally, the mass concentration of gluten in the gluten aqueous solution is 5% to 15%; Optionally, the ultra-high pressure homogenization conditions are: pressure 100-300 MPa, time 10-30 min; Optionally, the heating is heating the gluten aqueous solution to 40-60° C.; Optionally, the protease comprises: one of alkaline protease, neutral protease, composite protease, trypsin and papain; Optionally, the amount of the protease added is 0.5% to 1.5% of the mass of gluten; Optionally, the conditions for the enzymatic hydrolysis include: 40-60° C., 10-20 h.

8. A composition, characterized in that The active ingredients thereof include: the polypeptide according to claim 1, the polypeptide composition according to claim 2 or 3, or the gluten hydrolyzate prepared by the preparation method according to any one of claims 4 to 7; Optionally, the composition further comprises: brewer's yeast fermentation raw materials.

9. Use of the polypeptide according to claim 1, the polypeptide composition according to claim 2 or 3, or the gluten hydrolyzate prepared by the preparation method according to any one of claims 4 to 7 in yeast fermentation or preparation of yeast fermentation products; Optionally, the yeast comprises Saccharomyces cerevisiae.

10. Use of the polypeptide according to claim 1, the polypeptide composition according to claim 2 or 3, or the gluten hydrolyzate prepared by the preparation method according to any one of claims 4 to 7 in enhancing the resistance of yeast cells to salt stress or in preparing a product for enhancing the resistance of yeast cells to salt stress; Optionally, the yeast comprises Saccharomyces cerevisiae.

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